Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Summary

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Abstract

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Introduction

The retina, a vital visual sensory organ in humans and mammals, exhibits a distinct laminated structure characterized by three nuclear layers housing neuron somas and two plexiform layers formed by synaptic connections¹, including conventional synapses and the specialized ribbon synapse^2,3. The ribbon synapse plays a crucial role in transmitting vesicle impulses in a graded manner^2,3. The vision process involves electro-optical signal transmission across various levels of neurons and synapses, ultimately reaching the visual cortex^4,5.

Retinal organoids (ROs) represent a three-dimensional (3D) culture system derived from induced pluripotent stem cells (iPSCs), mimicking the physiological states of retinal tissue in vitro^1,6,7. This approach holds promise for studying retinal diseases⁸, drug screening⁹, and serving as a potential therapy for irreversible retinal degenerative conditions such as retinitis pigmentosa¹⁰ and glaucoma¹¹. As a powerful in vitro optical conduction system, the synapse within ROs is a crucial structure facilitating effective signal transformation and transfer⁵.

RO development can be roughly divided into three stages according to their morphological traits and molecular expression profiles^6,12. ROs in stage 1 (around D21-D60) comprise neural progenitor cells of the retina, many retinal ganglion cells (RGCs), and a few starburst amacrine cells (SACs), corresponding to the first epoch of human fetal development. In stage 2 (around D50-D150), ROs express some photoreceptor precursors, interneurons, and synaptogenesis-related genes, which represents a phase of transition. Photoreceptors develop maturity in stage 3 ROs (around after D100-D150), corresponding to the third stage of human fetal development^6,12,13. Notably, compared with ROs in stage 1 and stage 2, ROs in stage 3 have a distinct lamellar structure whose synapses have matured¹², including the presence of ribbon synapses¹⁴. Moreover, a recent study has confirmed that the mature synapses exist the transmission of light signals, indicating they are functional¹³. Thus, ROs in stage 3 are often selected to investigate synaptic structure.

Immunohistochemistry is widely applied to the study of the expression of various molecular proteins. However, the limitation of optical microscopy lies in its ability to observe only a restricted number of specific cells and molecules at a time, resulting in a lack of comprehensive analysis of the relationships between cells and their surrounding environment. Transmission Electron Microscopy (TEM) is characterized by high resolution, with a limited resolution of 0.1-0.2 nm, surpassing the light microscope by ~10-20 times¹⁵. It makes up for the defects of optical microscopy and is used to elucidate retinal development and synapse maturation in humans^16,17 and various species^18,19,20,21. TEM enables the direct distinction of presynaptic and postsynaptic components^18,20, and even allows for comprehensive observation of subcellular structures such as ribbons^2,3, vesicles²², and mitochondria²³. Therefore, TEM is an essential tool for identifying types of synapses and exploring the ultrastructure of synaptic contacts in ROs at the nanoscale.

It is crucial to note that sample preparation is of great importance for acquiring high-quality electron micrographs. Although some studies have performed EM on ROs^12,13,24, the detailed procedures are unclear. Since the quality of the electron microscopy image depends on the effect of RO fixation and reagent permeation to a large extent, various important factors need to be considered during preparation. Consequently, to better investigate synaptic contacts in ROs, we present a method with good reproducibility that shows the operation points of RO fixing, embedding, and the identification of observation sites.

Protocol

1. Obtaining ROs from iPSCs²⁵

NOTE: ROs were derived from iPSCs by modifying the previously reported procedure.

1. Dissociate iPSCs at ~90% confluence using a bacterial protease (see Table of Materials). Chop up the colonies into pieces and scrape them with a cell lifter.
2. After collection, resuspend the clusters of cells in 0.25 mL of ice-cold Matrigel. Following 20 min of incubation at 37 °C, scatter the solidified gel with the DMEM/F12 medium (0.5% N2 supplement, 1% B27 supplement, 0.1 mM β-mercaptoethanol, 0.2 mM L-glutamine substitute, 100 U/mL penicillin and 100 mg/mL streptomycin). Define this time as day 0 of differentiation.
3. After 5 days of floating culture, cells group to form cell cysts. Transfer these cell cysts into a 100 mm Petri dish where they will now adhere and grow.
4. On day 15, use bacterial protease to dissociate the adhering cells and culture them in DMEM/F12 medium (2% B27 supplement, 0.1 mM non-essential amino acids).
5. On day 21, change the medium with 20 mL of serum-containing medium (DMEM/F12 medium, 8% FBS, 2% B27 supplement, 100 mM Taurine, and 2 mM L-glutamine substitute), and replace the medium every week.

2. Anterior fixation of ROs

1. Gently move one of the cultured ROs (~D180, a size of about 1~2 mm in diameter is optimal) from the Petri dish to 1.5 mL microcentrifuge tubes with a pipette and mark the relevant information about the ROs.
   NOTE: Cut ROs in half or cut off the poorly structured tissue with a razor under the microscope in the dish containing fixative if the ROs are too large.
2. Remove the culture medium from 1.5 mL microcentrifuge tubes with a pipette. Add 1.5 mL of 2% paraformaldehyde-2% glutaraldehyde fixative diluted in 0.1 M phosphate buffer (PB), pH 7.4, and fix at room temperature (RT) for 4 h. Store the microcentrifuge tubes at 4 °C overnight.
   NOTE: This step can be paused for 1 week but restarting as early as possible is recommended. If ROs are left for longer, it may cause autolysis of cells leading to destruction of the ultrastructure of ROs. Due to the fragility of the ROs, avoid rotating, oscillating, flipping, or harsh operations.

3. Post fixation of ROs

1. Remove the 2% paraformaldehyde-2% glutaraldehyde fixative and wash with 1 mL of 0.01 M phosphate buffered saline (PBS) at pH 7.4 for 3 x 10 min. Gently suspend the ROs in the 1.5 mL microcentrifuge tubes during the washing process for fully rinsing. For every additional day of fixation, increase the number of washes by two to remove the residual fixative as much as possible.
2. Remove any remaining PBS using a pipette and replace it with ~150 µL of 1% osmic acid (OsO4) until the tissue blocks are submerged. Place the microcentrifuge tubes in a dark box to protect them from light and infiltrate at RT for 1 h.
   NOTE: OsO4 is a strong oxidant and must be handled in a fume hood.

4. Staining and dehydration

NOTE: The dehumidifier must be turned on to dry the environment from this step.

1. Remove OsO4 using a pipette, wash with 0.01 M PBS (pH 7.4) 3 x 10 min, and then wash 3 x 10 min with deionized water.
   NOTE: To ensure complete rinsing of ROs, we recommend suspending the ROs. Aspirate OsO4 into a dedicated disposal bottle.
2. Remove the last deionized water wash and replace with ~150 µL per tube (ensure full infiltration of tissue blocks) of uranium acetate and stain for 1-2 h at RT. Protect the microcentrifuge tubes from light.
3. Remove the uranium acetate and replace it with 1 mL of 50% acetone in the fume hood, dehydrating for 10 min. Then, use 70%, 80%, 90%, 100%, and 100% acetone for gradient dehydration of the ROs successively. Each dehydration lasts 10 min.
   NOTE: Leave some liquid to soak the ROs for each exchange to prevent exposure to air and moisture. Additionally, operate quickly as soon as possible because acetone is volatile. Aspirate uranium acetate into a dedicated disposal bottle.

5. Infiltration

1. Discard the acetone and add ~150 µL of the mixture consisting of acetone and Epon-812 resin at a ratio of 1:1. Place it in a 37 °C oven for 1 h.
2. Remove the above mixture and replace it with a mixture consisting of acetone and Epon-812 resin at a ratio of 1:4. Place it in a 37 °C oven overnight.
3. Transfer the ROs into the new tubes containing ~500 µL of pure epoxy resin carefully and place them in a 45 °C oven for 1 h.

6. Embedding

1. Prepare the 30-50 mL of pure epoxy resin in a 50 mL centrifuge tube by mixing the reagent kit according to the manufacturer's instructions in advance. Rotate the pure epoxy resin upside down slowly for 30 min to reduce air bubbles introduced by the mixer. Then, leave it in a 37 °C oven for 20 min. Add 2/3 volume of pure epoxy resin to the groove of the embedding mold.
2. Use a toothpick to pick ROs into the embedding mold equipped with pure epoxy resin.
3. Fill the groove with pure epoxy resin until it slightly protrudes or bulges.
4. Adjust the position of ROs at both ends of the embedding mold for directional embedding. Place the embedding mold in a 45 °C oven for 1-2 h and then put it in a 65 °C oven for 48-72 h.

7. Semi-thin positioning

1. Sand the excess epoxy resin around the tissue from the ends of the longer four sides of the embedding block to form a trapezoidal face. Install the embedding block into the sample mounting stand and screw down the bolt with an L screwdriver. Then, install glass knives on the knife stand and tighten the nut manually.
2. Trim away the excess resin until the desired area is exposed.
3. Fill the trough of the glass knife with water. Cut semi-thin slices to a thickness of 1 µm using a semi-thin microtome and lay them on a microscope slide with a needle.
4. Add 50 µL of 1% toluidine blue to dye semi-thin slices and incubate for 1 min at 95 °C on the heating plate. Rinse with distilled water for 2 min. Use a 20x ordinary optical microscope to observe the structure of the sections.

8. Ultra-thin sectioning

1. Install the embedding blocks and glass knives as described in step 7.1. Adjust the distance between the knife and the tissue block, the liquid level of the sink, and the slicing speed. Cut the ultrathin sections with a thickness of ~70-100 nm with an ultrathin microtome and scoop the slices onto the copper mesh (200 mesh) with the supporting film.
2. Stain ultrathin sections with 3% lead citrate for 15 min to enhance contrast in a dual staining procedure.

9. Imaging ROs by TEM

1. Launch the software (see the Table of Materials).
2. Click on the Memory mode button and mark each ultrathin section roughly.
3. Search for synaptic structures.
   1. Distinguish the laminated structure of ROs at low magnification.
   2. Locate the outer nuclear layer (ONL), which shows a nuclear structure with a deeper electron density. Subsequently, navigate to the terminal of the photoreceptor where numerous vesicles accumulate. Search for the rod terminals with a single synaptic ribbon and the cone terminals with multiple synaptic ribbons.
   3. Move to the inner plexiform layer (IPL) and seek out a similar structure with a small ribbon.
4. Capture the microscopic image.
   1. Click Start and input the corresponding magnifying power.
   2. Save the images.

Results

The establishment of 3D ROs through iPSC differentiation provides a powerful tool for studying retinal disease mechanisms and stem cell replacement therapy. Although others have demonstrated the synaptic connections in ROs functionally and immunocytochemically, direct evidence of conventional and ribbon synapses is very limited. Here we present a method for investigating the ultrastructure of two types of synapses in ROs by TEM. After 180 days of culture, ROs were fixed, stained, embedded, and ultrathin sliced. TEM obser...

Discussion

In this article, we presented a detailed protocol for observing conventional and ribbon synaptic ultrastructure in ROs by TEM. This protocol is based on the previously described retinal preparation methods with some modifications²⁰. To improve the success rate of sample treatment and the quality of TEM micrographs, consider the following key points. First, it is important to acknowledge that ROs develop from iPSCs, forming a cell mass lacking vasculature^6,

Materials

Name	Company	Catalog Number	Comments
100 mm Petri dish	Corning	430167
Acetone	Electron Microscopy Science	10000
B27 supplement	Gibco	A3582801
Cell lifter	Santa Cruz	sc-395251
Copper grids	Beijing Zhongjingkeyi Technology Co., Ltd.	AZH400HH
DigitalMicrograph Software	Gatan, Inc.		Software
Dispase	StemCell Technologies	#07913	Bacterial protease
DMEM/F12 medium	Gibco	#11320033
Embedding mold	Beijing Zhongjingkeyi Technology Co., Ltd.	GZ10592
Epon-812 resin	Electron Microscopy Science	#14900
Fetal Bovine Serum (FBS)	Biological Industries	#04-0021A
Glutaraldehyde	Electron Microscopy Science	16020
hiPSC	Shownin Biotechnology Co. Ltd.	RC01001-A
Lead citrate	Beijing Zhongjingkeyi Technology Co., Ltd.	GZ02618
L-GlutaMax	Life Technologies	#35050061	L-glutamine substitute
Matrigel	Corning	356234
Microscope slide	CITOTEST	80312-3161
N2 supplement	Gibco	17502048
Na2HPO4· 12H2O	Sigma	71650	A component of PB/PBS
NaH2PO4· H2O	Sigma	71507	A component of PB/PBS
Non-essential amino acids	Sigma	#M7145
Optical microscope	Lab Binocular Biological Microscope	Xsz-107bnii
OsO4	TED PELLA	4008-160501
Oven	Bluepard	BPG9040A
Paraformaldehyde	Electron Microscopy Science	157-8
Penicillin-Streptomycin	Gibco	#15140-122
Semi/ultrathin microtome	Reichert-Jung	396649
Taurine	Sigma	#T0625
Toluidine blue	Sangon Biotech	E670105-0100
Transmission Electron Microscopes	HITACHI	H-7500
Uranyl acetate	TED PELLA	CA96049
β-mercaptoethanol	Sigma	444203